2-Propenoic acid, 2-methyl-, tetracosyl ester, branched

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 December 2018 - 27 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenoic acid, 2-methyl-, tetracosyl ester, branched

EC Number:

292-146-9

EC Name:

2-Propenoic acid, 2-methyl-, tetracosyl ester, branched

Cas Number:

90552-24-2

Molecular formula:

C28H54O2

IUPAC Name:

2-Propenoic acid, 2-methyl-, tetracosyl ester(branched)

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. The Sprague-Dawley strain was selected as background data from previous studies are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) Males 10 wks; Females 11 wks
- Weight at study initiation: (P) Males: 432g to 477g, Females: 251 to 326g
- Housing: F0 animals were individually housed, except during mating (male + female) and lactation (female + pups), in polycarbonate cages (Tecniplast 2154, 48 x 26.5 x 21 cm, 940 cm2) with stainless steel lids and containing autoclaved sawdust.Toward the end of gestation and during lactation, females and their litters were provided with autoclaved wood shavings as nesting material. Each cage contained a rat hut and a piece of wood for the environmental enrichment of the animals.Cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): ad libitum access to SSNIFF rat/mouse pelleted maintenance diet, batch Nos. 14535074 and 94340868 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water (e.g. ad libitum): ad libitum access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: males 8 days before treatment, females 6 days before the beginning of estrous cycle monitoring during the pre-treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approx. 8 to 15 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Preparation procedure: According to Citoxlab France/Study No. 46857 VAS (homogeneity and stability testing) describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study.
Frequency of preparation: Based on test item dose formulation stability (Citoxlab France/StudyNo. 46857 VAS) and vehicle expiry.
Control dose formulation: According to the frequency of test item dose formulation preparation.
Storage and delivery conditions: At room temperature and protected from light. The stock solution was diluted with corn oil to achieve the concentration of the dosing solutions.

VEHICLE
- Justification for use and choice of vehicle: The vehicle for preparation of test item dose formulations was selected based on previous experimental work and/or preliminary studies.
- Concentration in vehicle: Solution from 3 to 300 mg/mL in the vehicle. Measured concentration = nominal concentration ± 10%.
- Amount of vehicle: A constant dosage volume of 5 mL/kg/day was used.
- Lot/batch no.: MKBX4739V

Details on mating procedure:

- M/F ratio per cage: One female was placed with one male
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: vaginal plug / sperm in a vaginal lavage referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individual housing of F0 pregnant animals. Near the end of gestation and during lactation, females and their litters were provided with autoclaved wood shavings as nesting material. Each cage contained a rat hut and a piece of wood for the environmental enrichment of the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of the dose formulations: Analytical technique: Gas Chromatography with Flame Ionization detection (GC-FID). Analytical method provided by the Sponsor and validated at Citoxlab France (Citoxlab France/Study No. 46857 VAS) prior to dose formulation analysis. Checked parameters, acceptance criteria and obtained results detailed in the validation report. The actual test item concentrations in the dose formulations were determined in Weeks 1, 3 and 6 using the validated GC-FID method. Acceptance criterion: Measured concentration = nominal concentration ± 10%. The actual test item concentrations in the analyzed dose formulations (i.e. in Weeks 1, 3 and 6) remained within an acceptable range of variation (-2.4% to +2.6%) when compared with the nominal values (nominal concentration ± 10% required).

Duration of treatment / exposure:

The dose formulations were administered daily according to the following schedule: Males: 2 weeks before mating, during the mating period (2 weeks), until euthanasia (at least 4 weeks in total).Females were treated for an overall period of 8 to 9 weeks:
2 weeks before mating, throughout mating (up to 2 weeks) and gestation (3 weeks), during lactation until Day 13 p.p. inclusive, until euthanasia for females with no delivery.

Frequency of treatment:

Daily

Details on study schedule:

Not applicable

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a previous study performed in the same species, wherein the test item was administered daily by gavage to five males and five females at dose levels of 0, 100, 300 or 1000 mg/kg bw/day for 2 weeks. The dose level of 1000 mg/kg bw/day was associated with ptyalism in one isolated female and with statistically significant higher or lower body weight gain in males or females, respectively,following initiation of treatment. This was followed by a higher body weight gain in females between Days 4 and 14 that correlated with higher food consumption from Day 4. Higher relative and absolute liver weights reached statistical significance in females and reduced size of testes and epididymides was observed in one male.The dose levels of 100 and 300 mg/kg/day were associated with ptyalism in isolated males and/or females, and a trend towards a slightly lower body weight change (statistically insignificant) was noted in females between Days 1 and 4. Therefore, 1000 mg/kg/day was selected as the high-dose level for the present study. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 100 and 300 mg/kg/day).
- Rationale for animal assignment: required number of animals (40 males and 40 females) were selected according to body weight and clinical condition, and allocated to groups (by sex) according to a stratified procedure based on body weight so that the average body weight of each group was similar. In addition, only females with regular estrous cycles were allocated to the groups (the regularity of estrous cycles monitored for days 14 days and confirmed 1 day before the beginning of the treatment period.
- Fasting period before blood sampling for clinical biochemistry: Overnight (minimum14 hours)
- Other: Identification: the animals (including supernumerary females) were identified by an individual ear tattoo. At the beginning of the study, each animal received a unique identity number.

Positive control:

Not Applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day before the treatment period and at least twice a day during the treatment period
- Cage side observations checked in table No.1- 4 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Performed on all animals once before the beginning of the treatment period and then once a week until the end of the study.
- Following parameters were examined:included (but were not limited to) changes in the skin, fur, eyes, mucous membranes,occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also evaluated

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each male recorded on the first day of treatment (Day 1), then once a week until euthanasia. Body weight of each female recorded on the first day of treatment (Day 1), then once a week until mated, and then on Days 0, 7, 14 and 20 p.c. (post-coitum), on the day of euthanasia for females that did not deliver and on Days 1, 4, 8 and 13 p.p. Females euthanized prematurely were weighed before euthanasia.

HAEMATOLOGY: Yes
- Time schedule: On the day of euthanasia
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: first five males and lactating females from each group
- Following parameters were examined: Erythrocytes (RBC), Mean cell volume (MCV), Packed cell volume (PCV), Hemoglobin (HB), Mean cell hemoglobin concentration (MCHC), Mean cell hemoglobin (MCH), Thrombocytes (PLT), Leucocytes (WBC), Differential white cell count with cell morphology (neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L),large unstained cells (LUC),monocytes (M)),Reticulocytes (RTC).

BLOOD CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of euthanasia
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: 14 hours
- How many animals: First five males and lactating females from each group
- Following parameters were examined: Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT),Total bilirubin (TOT.BIL), Total cholesterol (CHOL),Triglycerides (TRIG), Alkaline phosphatase (ALP), Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Bile acids (BIL.AC), Total proteins (PROT), Albumin (ALB), Albumin/globulin ratio (A/G)

THYROID HORMONES:
- Time schedulefor collection of blood: morning between 7.5 and 10 am
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: 14 hours
- How many animals: all animals

FUNCTIONAL OBSERVATION BATTERY:
-Time schedule for examinations: In each group, the first five males and lactating females that were euthanized as scheduled were evaluated with an FOB once at the end of the treatment period. For females, this was performed on Day 13 p.p. after euthanasia of the pups.

MOTOR ACTIVITY:
-Time schedule for examinations:automated infra-red sensor equipment over a 60-minute period.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue) each morning (between 7.5 and 10 am): during the 2 weeks of the pre-treatment period,from the beginning of the treatment period, during the pre-mating and mating periods, and until the females were mated, on the day of sacrifice, before euthanasia, to allow correlation with reproductive organ histopathology data.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 pups/litter 4/sex/litter as nearly as possible; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, presence of gross anomalies, weight gain, physical abnormalities, anogenital distance (AGD), pup weight on the day of AGD, mortality and clinical signs, presence of nipples/areolae in male pups, sex ratio (% of males) thyroid hormone levels. Particular attention was paid to the external genital organs and to whether the pup had been fed (e.g. presence of milk in the stomach) when possible. Then they were discarded without any further examination.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE: On completion of the treatment period, after at least 14 hours fasting, all surviving F0 animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination
- Male animals: All surviving animals after the end of the mating period
- Maternal animals: All surviving animals on Day 14 p.p. Female P22813 (group 4) with total litter loss was euthanized on Day 2 p.p. Female P22786 (group 2), which did not deliver, was euthanized on Day 26 p.c.

Animals prematurely euthanized (F0 Females)
During the gestation period:
prematurely euthanized female P22785 (group 2) was euthanized by inhalation of carbon dioxide gas followed by cervical dislocation and a complete macroscopic post-mortem examination was performed. The pregnancy status was determined and the number of corpora lutea and implantation sites wasrecorded and classified as live or dead concepti, early or late resorptions or scars.
During delivery:
Female P22806 (group 3) had difficulty to deliver and was euthanized by inhalation of carbon dioxide gas followed by cervical dislocation and then a complete macroscopic post-mortem examination was performed. The total number of corpora lutea, implantation sites, live and dead fetuses/pups and early or late resorptions was recorded.
During the lactation period:
female P22813 (group 4) was euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination before being submitted for a complete macroscopic post-mortem examination. In addition, the number of corpora lutea and implantation sites was recorded

ORGAN WEIGHTS: The body weight of each F0 animal euthanized as scheduled (after the end of the mating period for males or on Day 14 p.p. for females) was recorded before euthanasia. For these animals, the organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection, or after fixation for thyroids with parathyroids (if applicable).

GROSS NECROPSY
- Gross necropsy consisted of a complete macroscopic post-mortem examination of all F0 animals, including those prematurely euthanized. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 14 p.p. and for female P22786 (group 2) euthanized on Day 26 p.c. due to no delivery.

HISTOPATHOLOGY
All tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable, embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes
and epididymides which were stained with hematoxylin/PAS).The tissues indicated in Tissue procedure table were prepared for microscopic examination and weighed, respectively. A microscopic examination was performed on: all tissues listed in the Tissue Procedure Table from the first five euthanized as scheduled males and lactating females in the control and high-dose groups (groups 1 and 4), all macroscopic lesions (all groups), all tissues listed in the tissue Procedure Table from female Nos. P22806 (group 3) and P22813 (group 4) that were euthanized prematurely, reproductive organs and thyroids from pregnant female P22786 (Group 2) that did not deliver, to investigate possible causes.Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Reference: (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004)

Postmortem examinations (offspring):

SACRIFICE: Pups were euthanized by an intraperitoneal injection of sodium pentobarbital or by decapitation under isoflurane anesthesia on Day 4 p.p. when blood was collected, followed by exsanguination when the thyroids were sampled. Animals prematurely euthanized: Moribund pups were euthanized by an intraperitoneal injection of sodium pentobarbital. Found dead and prematurely euthanized pups were submitted to a detailed external examination (including orifices and buccal cavity), after euthanasia when applicable.Pups not selected on Day 4 p.p. were discarded without further examination. Pups euthanized on Day 13 p.p. were submitted to a detailed external examination (including orifices and buccal cavity) after euthanasia. Particular attention was paid to the external genital organs. Then the animals were discarded without any further examination, or after collection of thyroids with parathyroids for selected pups.
- The F1 offspring not selected as parental animals were sacrificed at: pups not selected on Day 4 p.p.,surviving pups: on Day 13 p.p.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: Organ Weights: The body weight of one selected pup/sex/litter euthanized on Day 13 p.p. was recorded before euthanasia. Thyroids with parathyroids from the selected pup/sex/litter euthanized on Day 13 p.p. were preserved in 10% buffered formalin


GROSS NECROPSY: Pups euthanized on Day 13 p.p. were submitted to a detailed external examination (including orifices and buccal cavity) after euthanasia.

Statistics:

Body weight, food consumption and reproductive data:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
Hematology, blood biochemistry, hormones, motor activity, anogenital distance, nipples/areolae, live birth index(es), sex ratio and post-implantation loss: CITOX software was used to perform the statistical analysis of these data
Organ weight: PATHDATA software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01)

Reproductive indices:

Mating index: (Number of mating animals/Number of paired animals) x 100
Fertility index:(Number of pregnant female partners/ Number of mated pairs) x 100
Gestation index:(Number of females with live born pups/Number of pregnant females) x 100
pre-implantation loss: (Number of corpora lutea - Number of implantation sites/Number of corpora lutea) x 100

Offspring viability indices:

Live birth index:(Number of live born pups on Day 1 p.p./Number of delivered pups)
Viability index: (Number of surviving pups on Day 4 p.p. (before culling) / Number of delivered pups) x 100
Lactation index: (Number of surviving pups on Day 13 p.p./Number of surviving pups on Day 4 p.p. (after culling)) x 100
post-implantation loss (calculated with Excel): (Number of implantation sites - Number of live pups/Number of implantation sites) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Ptyalism observed on a few occasions in one isolated male at 300 and 1000 mg/kg/day, in one isolated female at 300 mg/kg/day (during the gestation period) and in one isolated female at 1000 mg/kg/day (during the lactation period). This sign is commonly noted when a test item is administered by gavage. All the other clinical signs (i.e. areas of hair loss, cutaneous lesions, scabs, chromodacryorrhea, chromorhinorrhea and/or short/broken teeth) considered to be unrelated to the test item as they were present in control animals, were not dose-related, were reported sporadically in only a few animals, are commonly observed findings in this species and strain.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths in control males or females. In females, there were the following unscheduled deaths: Gestation period: At 100 mg/kg/day: Female P22785 was euthanized on Day 19 p.c. for humane reasons as a detectable mass was observed in the urogenital region which would have impeded delivery. At necropsy, a subcutaneous homogenous mass (firm in consistency, measuring approximately 2.5 cm in diameter) was noted in the urogenital region. This mass was a mammary adenocarcinoma. This type of tumor is occasionally observed in females of this age, and displayed no relationship to test item administration. This female was pregnant with 19 live fetuses. Female P22786 was euthanized on Day 26 p.c. for absence of delivery. No extraordinary clinical signs were observed prior to euthanasia. At necropsy, the uterus was dilated and reddish in color. In the absence of relevant microscopic changes on genital organs, this absence of delivery is considered spontaneous and unrelated to test item administration.This female was pregnant with one dead fetus and its corresponding placenta. At 300 mg/kg/day Female P22806 was euthanized on Day 24 p.c. due to delivery difficulties. Signs of poor clinical condition i.e. pallor of extremities, piloerection and round back) were observed prior to euthanasia. Eight dead or cannibalized pups were found in the bedding, and five dead fetuses were noted in the uterus. At necropsy, the thymus had a gelatinous appearance; the lungs, liver and stomach wall showed tan or reddish discoloration, and the vagina had reddish liquid content. Finding in the liver correlated with moderate centrilobular necrosis. This is interpreted as evidence of chronic passive congestion, probably as a consequence of dystocia. This isolated dystocia at the intermediate dose only is considered unrelated to test item administration, since this can occasionally be observed in control dams. Lactation Period: At 1000 mg/kg/day Female P22813 was euthanized on Day 2 p.p. as her litter was found dead (10 pups at birth). Two pups were found dead on Day 1 p.p and eight cannibalized or dead pups were found in the bedding on Day 2 p.p. No remarkable clinical signs were observed prior to euthanasia. At necropsy, two placentae and 14 implantation sites were recorded in the uterus. In the absence of relevant microscopic changes, it is assumed that the death of the litter was related to an absence of maternal behaviour most probably due to the presence of placentae in the uterus. As this finding is isolated and can occasionally be observed in control dams, this was considered to be unrelated to test item administration.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related differences in mean body weight or mean body weight change at any dose level in males or females during the premating, gestation and lactation periods at any dose level. When compared with controls, a slight mean body weight loss was recorded in females given 1000 mg/kg/day at the beginning of the lactation period (-3 g vs. +2 g). This was followed by a higher mean body weight gain during the second recording interval (+18 g vs. +12 g). This difference was not statistically significant, did not impact the mean body weight or the mean body weight gain over the whole lactation period. Therefore these findings were considered to be non adverse.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects on mean food consumption in males or females. Differences between control and test item-treated animals consisted of slightly higher mean food consumption during: the mating period between Days 1 and 8 in females given 100, 300 or 1000 mg/kg/day (+28%, +17% and +17%, respectively) and between Days 8 and 15 in females at 100 and 1000 mg/kg/day (+33% and +39%, respectively), the whole gestation period in females given 100 (up to +19%; p< 0.001 between Days 7 and 14 p.c.) or 1000 mg/kg/day (up to +33%; up to p<0.01) and in females given 300 mg/kg/day (between Days 7 and 14 p.c.: +8%, p<0.05),the lactation period between Days 1 and 8 p.p. in females given 100 mg/kg/day (+12% to +18%) and between Days 1 and 4 p.p. in females given 300 mg/kg/day (+18%). A relationship with the test item was considered to be unlikely as these variations were poorly dose-related, due to isolated individual values (i.e. group 2 females P22784 and P22790 during the premating period,group 4 female P22810 for which spillage was observed throughout the first week of the premating period and group 4 females P22812 and P22813 during the gestation period) and/or there were no correlation with mean body weight change.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on hematology parameters at any dose level. All the differences from controls, namely lower mean platelet count in males and/or females at all dose levels, lower mean neutrophil and/or basophil counts in males and/or females at all dose levels, lower mean lymphocyte count in females at all dose levels, lower mean large unstained cell count in females given 100 or 300 mg/kg/day, lower or higher mean monocyte count in males or females given 1000 mg/kg/day and/or higher mean eosinophil count in males given 100 mg/kg/day, were of low magnitude, of opposite trends, due to the presence of platelet aggregates (artefacts at 100 mg/kg/day), unrelated to microscopic findings and/or poorly dose-related. Therefore, these differences were considered to be unrelated to the test item treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day and when compared with controls, statistically significant, dose-related, higher mean chloride level was noted in males (+1%). In the absence of similar finding in the opposite sex and any correlation with microscopic observations and/or in view of the low magnitude, this difference was considered to be of no toxicological importance. The other differences from controls consisted of low mean total bilirubin and/or low mean biliary acid levels in males and/or females at all dose levels, low mean cholesterol levels in females given 100 or 1000 mg/kg/day and/or high mean triglyceride level in females given 300 mg/kg/day. In view of the low magnitude and the directions of the changes, the absence of related microscopic findings and/or as mean values were not dose-related, these findings were not considered to be toxicologically important.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor Activity: There were no relevant differences between the test item-treated and control groups in motor activity (horizontal movements and rearing) in males or females. Differences from controls were noted in the mean number of horizontal and rearing movements at 100 mg/kg/day (males: +18% and females: -30%, and females: -32%, respectively), at 300 mg/kg/day (males: -15% and females: -28%, and females: -26%, respectively) and in the mean number of horizontal movements at 1000 mg/kg/day (males: +13%).These values were considered to be of no toxicological importance as they were of opposite trends, not statistically significant, poorly dose-related and/or as values remained close to or within the standard deviation of control values. Functional Observation battery: An absence of grooming was recorded in 5/5 females at 100 mg/kg/day (versus 4/5 in control females) and in 5/5 males at 1000 mg/kg/day (versus 3/5 in control males). An absence of urination was noted in 5/5 females at 1000 mg/kg/day (versus 3/5 in control females). Higher mean landing foot splay was recorded in males given 300 or 1000 mg/kg/day (+16% and +18% vs. controls, respectively) whereas lower mean values were recorded in females given 100 or 300 mg/kg/day (-16% and -11% vs. controls, respectively).These findings were considered to be of no toxicological importance as they were only observed in one sex, were poorly dose-related and/or did not correlate with any other findings.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All observations belonged to the spectrum of spontaneous changes in rats of this age and strain and bore no relationship to test item administration. In particular, there were no test item treatment related changes in the reproductive organs.
Two females at the high dose had started cycling (estrus or diestrus). Vaginal mucification was observed in other high dose and in controls dams, as expected for lactating females (pups starting to eat solid food from Day 14 p.p.). During this transitory period, diestrus or estrus stages are also expected to be observed in a few dams that have started cycling.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Gestation period: At 100 mg/kg/day Female P22785 was euthanized on Day 19 p.c. for humane reasons as a palpable mass was observed in the urogenital region that would have impeded delivery. At necropsy, a subcutaneous homogenous mass (firm in consistency, measuring approximately 2.5 cm in diameter) was noted in the urogenital region. This mass was a mammary adenocarcinoma.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The thyroid hormone levels were considered to be unaffected by the test item in F0 males. When compared with controls, there was a slightly higher mean T4 concentration at 1000 mg/kg/day. Higher mean TSH concentrations were also noted in all test item-treated groups. As these differences did not correlate with each other, were of minor magnitude, not statistically significant, poorly dose-related, and/or due to isolated values and/or as individual vales remained within the range of control values, they were considered to be of no toxicological importance.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No effects on the mean length of the estrous cycle or the mean number of cycles.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects on pairing, mating or fertility indexes. High mean pre-coital time was recorded in controls. This was mainly due to one isolated pair (female P22777 and male P22477) that mated on Day 13 of the mating period. There were no effects on the mean duration of gestation, numbers of corpora lutea and implantation sites, numbers of pups delivered or percentages of pre-/post-implantation loss.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive performance

2

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no external abnormalities at pup examination .At 1000 mg/kg/day and when compared with controls, an increased number of clinical signs due to a lack of maternal care (generalized pallor, coldness to the touch, slow breathing, thin appearance, dehydration, absence of milk in the stomach and/or hematoma on the back) was noted in pups. This was mainly due to one litter (female P22813) for which the pups were found dead or cannibalized on Day 1 or 2 p.p., but a test item relationship could not be excluded as an increased incidence of affected (litters 4/10 excluding litter P22813 vs. 1/10 in controls) was recorded. As the viability index and the mean body weight of pups were not affected, these findings were not considered to be adverse. At 300 mg/kg/day, generalized pallor was observed in one pup. As this clinical sign was isolated and only observed transiently, it was considered to be incidental. Other clinical signs (i.e. findings on forelimbs and/or hindlimbs, tail and/or anal region, hematoma on the head, and/or wound on the back and/or abdomen) were not attributed to the test item as they resulted from the ink injection performed to identify pups and/or were also reported in controls.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on viability. At 1000 mg/kg/day, there was an increase in the number of found dead, prematurely sacrificed or cannibalized pups (20 pups vs. 14 control pups). This did not impact the incidence of affected litters (5 litters vs. 6 litters) or the mean live birth index and it resulted from the unrelated test item death of 10 pups of female P22813 (on Days 1 and 2 p.p.).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no relevant effects on mean body weight or mean body weight change during the lactation period. On Day 1 p.p. and when compared with controls, statistically significant higher mean body weight was recorded in male and female pups at 300 mg/kg/day (+9% and +12%, respectively). A relationship with the test item was considered to be unlikely as these variations were not dose-related and were of minor amplitude.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no test item-related effects on AGD and AGD/BW in the pups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples or areolae were observed in any male pups.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on sex ratio (% of male pups).The thyroid hormone levels were considered to be unaffected by the test item in pups.There was lower mean TSH concentrations (not statistically significant) in pups at 100 mg/kg/day when compared to controls These differences were considered as fortuitous in the absence of a dose-response relationship and as there was no correlation with any other findings.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

sexual maturation

clinical signs

mortality

body weight and weight gain

other: Thyroid hormones

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The test item, 2-Propenoic acid, 2-methyl-, tetracosyl ester (branched), was administered daily by oral gavage to male and female Sprague-Dawley rats for 2 weeks before mating, during mating and, for females, throughout gestation and until Day 13 p.p. at the dose levels of 100, 300 or 1000 mg/kg/day.

Based on the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 1000 mg/kg/day (high-dose level) in the absence of adverse findings,
- the No Observed Effect Level (NOEL) for reproductive performance (mating, fertility and delivery) was considered to be 1000 mg/kg/day in the absence of any test item-related findings,
- the No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was considered to be 1000 mg/kg/day in the absence of adverse findings.

Executive summary:

A GLP compliant study assessing the potential toxic effects of 2- Propenoic acid, 2-methyl-, tetracosyl ester (branched) henceforth referred to as “test item” was conducted using the OECD guideline 422: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test method. The test item was administered to three groups of 10 male and 10 female Sprague-Dawley rats, daily, by the oral route (gavage) at dose levels of 100, 300 or 1000mg/kg/day, using a constant dosing volume of 5ml/kg/day. Males were treated for an overall period of at least 4 weeks (2 weeks before mating, during the mating period and until the day before euthanasia). Females were treated for an overall period of 8 to 9 weeks (2 weeks before mating, throughout mating (up to 2 weeks) and gestation (3 weeks) until day 13 post-partum (p.p.) inclusive). A control group consisting of 10 male and 10 females received the vehicle (peanut oil) alone under the same experimental conditions. Concentrations of the test item in the dose formulations were determined using a validated Gas chromatography with flame ionisation detection analytical method in weeks 1, 3 and 6. F0 (Parental) animals were observed daily during the treatment period for mortality, morbidity and clinical signs. No test item-related unscheduled deaths were noted, in relation to clinical signs, ptyalism occurred in one isolated male at 300 and 1000 mg/kg/day and in one isolated female at 300 and 1000 mg/kg/day during the gestation or the lactation period, respectively. This sign was not considered as an adverse effect as it is frequently detected when a test item is administered by gavage. Comprehensive clinical observations were conducted on a weekly basis. Body weights and food consumption were recorded weekly during the premating, mating, gestation and lactation periods (food consumption not recorded during mating period). No test-item related effects on mean body weight or body weight change at any dose level were noted. Estrous cycle stage was determined each morning from 2 weeks before the treatment period until the females had mated, and on Day 14 p.p. before euthanasia. No effects on the mean length of the estrous cycle or the mean number of cycles at any dose level were noted. The F0 animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until Day 13 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. No test item related effects were observed on the mating, fertility, gestation indexes, mean duration of gestation, numbers of corpora lutea, implantation sites, numbers of pups delivered, percentages of pre-/post-implantation losses or mean percentage of male foetuses (sex ratio) at any dose level. No relevant effects on the live birth, viability or lactation indexes at any dose level were noted for the pups. The size of each litter was adjusted on Day 4 p.p. by culling extra pups to obtain close to four males and four females per litter. Pups not selected on Day 4 p.p. were euthanized and discarded without further examination. Selected pups were weighed on Days 1, 4, 8 and 13 p.p. and observed daily for clinical signs, abnormal behaviour and external abnormalities. No test item-related effects on mean body weight, mean body weight change or external abnormalities in pups at any dose level were found. In the pups at 1000 mg/kg/day, there were additional clinical signs such as: generalized pallor, coldness to the touch, slow breathing, thin appearance, dehydration, absence of milk in the stomach and/or hematoma on the back, when compared with controls. This was postulated to be due to a deficiency in maternal care and more litters were affected (5/10 vs. 1/10 in controls) findings were not considered to be adverse as there were no effects on the viability and the weight of pups. The physical development of pups was assessed by measuring the anogenital distance on Day 1 p.p. and by counting the number of nipples and areolae in male pups on Day 12 p.p. No test item-related effects were noted on the anogenital distance and there were no nipples or areolae in male pups at any dose level. A Functional Observation Battery (FOB) was performed on five F0 animals per sex and group at the end of the treatment period where no test item-related effects on FOB or motor activity data at any dose level were noted. Laboratory investigations (haematology and blood biochemistry) were carried out on designated F0 males and F0 females from each group at the end of the study. No test item-related effects on haematology parameters or blood chemistry at any dose level were noted. Plasma thyroid hormone levels (TSH and T4) were determined at termination in all F0 males and in pups euthanized on Day 13 p.p. No test item-related effects on T4 or TSH levels in F0 males or in pups were found. The F0 males were euthanized after completion of the mating period (i.e. after 30 days of treatment) and F0 females were euthanized on Day 14 p.p. A full macroscopic post-mortem examination was performed on F0 animals, with particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from five males and lactating females in the control and high-dose groups, in one group 3 female and one group 4 female that were prematurely euthanized and on all macroscopic lesions (all groups). The test item was not associated with any organ weights, macroscopic or microscopic changes. Pups were euthanized on Day 13 p.p. and a detailed external examination was performed, with particular attention to the external genital organs no test item-related effects at any dose level were observed.

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was found to be 1000 mg/kg/day (high-dose level) in the absence of adverse findings, the No Observed Effect Level (NOEL) for reproductive performance (mating, fertility and delivery) was found to be 1000 mg/kg/day in the absence of any test item-related findings. Finally, the No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was considered to be 1000 mg/kg/day in the absence of adverse findings.